Staphylococci causing hospital-acquired infections are increasingly resistant to multiple antimicrobial agents. The trait that defines the multidrug-resistant phenotype is resistance to beta-lactam or methicillin resistance (MR). The chromosomal gene responsible for MR, mecA, encodes a penicillin binding protein with low beta-lactam affinity. MecA is found only in resistant isolates, is common to all resistant staphylococcal species and is accompanied by >30 kb of DNA (mec DNA) that is also unique to resistant isolates. Contained within the mec DNA, just 5' to mecA, are two genes, mecRI and mecI, that regulate the transcription of mecA. This proposal will investigate the regulation of mecA by mecRI and mecI; the contribution of regulation to phenotype; and the mutations, insertions, deletions and rearrangements of mec-associated DNA that alter regulatory genes in clinical isolates. In addition, the organization and extent of the entire mec locus will be determined in three staphylococcal isolates. The regulatory loci will be cloned by PCR from wild isolates of defined phenotype and introduced into a MR S. aureus strain with deleted regulatory genes. The contribution of intact and altered regulatory genes to phenotypic resistance and mecA expression will be investigated; induction will be studied by analyzing mRNA transcripts and the PBP2A gene product; the specific role of mutations in determining phenotype will be assessed by making mecR1-mecI fusions; and the failure of reporter gene induction will be used to locate additional inactivated chromosomal regulatory loci. The total extent and composition of mec DNA will also be determined among MR S. aureus and coagulase-negative staphylococci by cloning large, overlapping DNA fragments and DNA sequencing and PCR amplification, generating probes that can be used to compare mec restriction fragment length polymorphisms among a large number of clinical MR isolates. These studies will help to define the role of specific mec-associated DNA in the expression of resistance; identify other regulatory circuits involved in signal transduction; further assess the clonality of MR staphylococci and identify specific DNA sequences that can be used for assessing the molecular epidemiology of hospital-acquired staphylococci.